NR ACWA
AU Cooley,W.A.; Davis,L.A.; Keyes,P.; Stack,M.J.
TI The reproducibility of scrapie-associated fibril and PrPsc detection methods after long-term cold storage of natural ovine scrapie-affected brain tissue
QU Journal of Comparative Pathology 1999 May; 120(4): 357-68
PT journal article
AB A pool of grey matter (medulla/brain stem, cerebellum and frontal cerebral cortex) was prepared from the brains of 16 sheep with scrapie, diagnosed clinically and by the demonstration of spongiform encephalopathy. Aliquots from the pool of tissue were finely chopped or homogenized and stored at +4 degrees C or -70 degrees C, after undergoing one of several specific pre-treatments (storage with or without protease inhibitors or, alternatively, with or without the cryoprotectant, dimethyl sulphoxide). At intervals over a period of 2 years, the stored extracts were examined by electron microscopy for the presence of scrapie-associated fibrils (SAFs) and by Western immunoblotting for the disease-specific abnormal protein PrPsc. Throughout the 2-year period, SAFs and PrPsc were detected in the majority of all stored tissue extracts under all combinations of tissue preparation and pre-treatment. The combined detection rates for SAFs and PrPsc were 91% at +4 degrees C and 94% at -70 degrees C. There was no significant difference between the results obtained by the two detection methods and no specific combination of preparation method and pre-treatment was superior to any other. Storage of the samples at -70 degrees C appeared to give better results than storage at +4 degrees C, particularly with regard to fibril detection. For logistical reasons and ease of processing, and to avoid the effects of autolysis on recognizable brain regions, long-term storage at -70 degrees C, without any pre-treatment, would appear to be the method of choice.
IN Ein Pool aus der grauen Hirnsubstanz von 16 Schafen mit klinisch und histologisch diagnostiziertem Scrapie wurde in kleinen Fraktionen fein zerhackt oder homogenisiert, mit oder ohne Proteaseinhibitoren, mit oder ohne DMSO aus Gefrierschutz bei +4° oder bei -70° bis zu 2 Jahre lang gelagert. Während dieser 2 Jahre wurden wiederholt einige der unterschiedlich behandelten Proben aufgetaut, um entweder elektronenmikroskopisch die scrapieassoziierten Fibrillen oder per Western blot die proteaseresistenten Prionproteine nachzuweisen. Dies gelang bei 91% der bei +4° und sogar bei 94% der bei -70° gelagerten Proben unabhängig von der Vorbehandlung und dem Nachweisverfahren. Daher wird eine Langzeitlagerung bei -70° ohne Vorbehandlung empfohlen.
ZR 41
MH Animal; Blotting, Western; Brain/*ultrastructure; Brain Chemistry; *Cryopreservation; Microscopy, Electron; PrP 27-30 Protein/*ultrastructure; PrPsc Proteins/*analysis; Predictive Value of Tests; Reproducibility of Results; Scrapie/*pathology; Sheep; Support, Non-U.S. Gov't
AD Veterinary Laboratories Agency, Central Veterinary Laboratory, Woodham Lane, New Haw, Weybridge, Surrey, KT15 3NB, UK
SP englisch
PO England